KR101538468B1 - Skin external composition for enhancing skin barrier or moisturizing skin containing 7,3',4'-trihydroxyisoflavone - Google Patents

Abstract

The present invention relates to a composition for external application for skin barrier strengthening or moisturizing skin containing 7,3 ', 4'-trihydroxyisoflavone as an active ingredient, 3 ', 4 ' -trihydroxyisoflavone to activate and activate the peroxisome proliferator activated receptor alpha (PPAR [alpha]) in vivo, as well as to increase expression of the pilar green gene, transglutaminase 1 gene, The present invention relates to a composition for external application for skin, which increases expression of hyaluronic acid synthase 2 gene, induces differentiation of keratinocytes, strengthens skin barrier, and increases skin moisture content.

7,3 ', 4'-trihydroxyisoflavone, skin barrier enhancement, skin moisturizing, peroxisome proliferator activated receptor, pilar green, transglutaminase, hyaluronic acid synthase

Description

[0001] The present invention relates to a composition for skin barrier enhancement or moisturizing skin containing 7,3 ', 4'-trihydroxyisoflavone containing 7,3', 4'-trihydroxyisoflavone,

The present invention relates to a composition for external application for skin barrier strengthening or moisturizing skin containing 7,3 ', 4'-trihydroxyisoflavone as an active ingredient, 3 ', 4 ' -trihydroxyisoflavone to activate and activate the peroxisome proliferator activated receptor alpha (PPAR [alpha]) in vivo, as well as to increase expression of the pilar green gene, transglutaminase 1 gene, The present invention relates to a composition for external application for skin, which increases expression of hyaluronic acid synthase 2 gene, induces differentiation of keratinocytes, strengthens skin barrier, and increases skin moisture content.

The skin is largely divided into three layers: epidermis, dermis, and subcutaneous tissue in order from the outside. It has a function to protect the internal organs of the body from changes in temperature and humidity, ultraviolet rays, and other physical and chemical external stimuli. In particular, the epidermis plays an important role in preventing water evaporation inside the human body.

The epidermis is divided into the stratum corneum, granular layer, the superficial layer and the basal layer in order from the outside, and the cells of the stratum corneum act as bricks and the intercellular lipids between the keratinocytes function as the mortar to constitute the skin barrier J. Invest. Dermatol 80 (Suppl.), 44-49, 1983). In addition, a healthy human keratinocyte has a high concentration of natural moisturizing factor (NMF) to help maintain moisture in the skin. For example, substances such as amino acids are water-soluble, (J. Invest. Dermatol. 54, 24-31, 1970).

However, as it is nowadays, there is a tendency to change the temperature and humidity of the environment due to changes in the environment and life patterns, anthropogenic temperature control of the heating and cooling, skin stress caused by various stresses and environmental pollution in the social life, Skin aging and the like, the moisture of the stratum corneum is decreased, and the skin becomes dry, the surface becomes rough, the skin becomes loose, the moisture is lost, and the appearance of lack of vitality occurs. Therefore, composition for skin barrier strengthening or skin moisturizing Is increasing.

Peroxisome proliferator-activated receptors (PPARs) play an important role in regulating cell proliferation / differentiation, regulation of inflammatory mediators, and also play an important role in the regulation of glucose, lipid and hormone metabolism (Kuenzli, S., Saurat, JH), Peroxisome proliferator-activated receptors in cutaneous biology, Br. J. Dermatol., 149 (2003), 229-236; Desvergne, B., Wahli, W., Peroxisome proliferator-activated receptors: nuclear control of metabolism, Endocrine Reviews, 20 (1999) 649-688.). In particular, it has been found that the epidermis of the skin promotes keratinocyte differentiation, suppresses proliferation, promotes skin barrier formation through lipid metabolism, inhibits inflammation and plays an important role in maintenance of homeostasis of skin function.

Isoflavones are mainly vegetable compounds contained in soybean, and they exist as glucosides containing isoflavones as aglycon. They are converted into non-saccharide form by metabolism of microorganisms during fermentation. These isoflavones have been known to have anticancer, antioxidant, antiarteriosclerosis, hypoglycemic and osteoporosis prevention effects, and studies have been conducted actively.

Accordingly, the inventors of the present invention have investigated the effect of ortho-dihydroxyisoflavone and found that 7,3 ', 4'-trihydroxyisoflavone activates peroxisome proliferator activated receptor alpha (PPAR alpha) It regulates the expression of keratinocytes, induces the differentiation of skin cells, strengthens the skin barrier, and promotes the skin moisturizing function of the natural protein moisturizing factor Pillagreen gene, which plays a crucial role in the formation of skin keratinization membrane transglutaminase 1 Gene, and hyaluronic acid synthase 2 gene, which is responsible for the synthesis of hyaluronic acid, which is an extracellular matrix component that performs skin moisture-retaining function, and thus completed the present invention.

Accordingly, an object of the present invention is to provide a composition for external application for skin, which comprises 7,3 ', 4'-trihydroxyisoflavone and has an effect of inducing differentiation of skin epidermis, a skin moisturizing effect and a skin barrier strengthening effect.

In order to accomplish the above object, the present invention provides a composition for external application for skin barrier comprising 7,3 ', 4'-trihydroxyisoflavone as an active ingredient.

Also, the present invention provides a composition for external application for moisturizing skin containing 7,3 ', 4'-trihydroxyisoflavone as an active ingredient.

The composition for external application for skin according to the present invention contains 7,3 ', 4'-trihydroxyisoflavone as an active ingredient to induce PPARs transcription activity or increase expression thereof, thereby inducing differentiation of keratinocytes and strengthening skin barrier To increase skin moisturization. In addition, the composition for external application for skin according to the present invention is characterized in that it comprises a filaggrin gene which is a precursor protein of a natural moisturizing factor responsible for moisturizing the skin surface, a transglutaminase 1 gene which plays a crucial role in the formation of a skin keratinization film, The skin moisturizing effect and the skin barrier enhancement effect by increasing the expression of the hyaluronic acid synthase 2 gene responsible for the synthesis of hyaluronic acid, which is an extracellular matrix component that performs the skin epithelium differentiation.

The present invention provides a composition for external application for skin for enhancing skin barrier comprising 7,3 ', 4'-trihydroxyisoflavone as an active ingredient.

The present invention also provides a moisturizing external preparation for skin comprising 7,3 ', 4'-trihydroxyisoflavone as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The 7,3 ', 4'-trihydroxyisoflavone used as an active ingredient in the present invention is an ortho-dihydroxyisoflavone (ODI) Is higher than other isoflavones.

The 7,3 ', 4'-trihydroxyisoflavone of the present invention is represented by the following formula (1).

The 7,3 ', 4'-trihydroxyisoflavone of the present invention induces transcription activity of peroxisome proliferator activated receptor alpha (PPAR alpha) or induces differentiation of keratinocytes by increasing its expression, Strengthens the barrier and increases skin moisturization. In addition, the filaggrin gene, which is a precursor protein of the natural moisturizing factor (NMF) responsible for moisturizing the skin surface, transglutaminase 1, which plays a crucial role in the formation of a corneal envelope (Hyaluronic acid synthase 2, HAS 2) gene, which is responsible for the synthesis of hyaluronic acid, which is a transglutaminase 1, TGase 1) gene and an extracellular matrix component that performs skin moisture retention function, A skin moisturizing effect, and a skin barrier strengthening effect.

The 7,3 ', 4'-trihydroxyisoflavone of the present invention can be produced by biotransformation of a daidzein by a well-known method well known in the art, but not limited thereto, Production is possible.

It is preferred that the 7,3 ', 4'-trihydroxyisoflavone of the present invention is contained in an amount of 0.001 to 10% by weight based on the total weight of the composition. If the content is less than 0.001% by weight, the effect of inducing the skin epidermal differentiation inducing effect, skin moisturizing effect and skin barrier strengthening effect can not be obtained, and if it exceeds 10% by weight, the increase in the effect is not so large compared with the increase in the content.

The composition for external application for skin according to the present invention may be prepared in the form of a pharmaceutical composition containing an effective amount of 7,3 ', 4'-trihydroxyisoflavone, and may contain one or more non-toxic, A pharmaceutically acceptable carrier, adjuvant or diluent or other active ingredient.

The composition for external application for skin according to the present invention may be formulated by a known method using a pharmaceutically acceptable carrier and an excipient.

In addition, the composition for external application for skin according to the present invention may be in the form of a solution, a suspension or an emulsion in an oil or an aqueous medium, or may be formulated in the form of a dry powder to be dissolved in sterile, exothermic water before use. A water-in-oil type emulsion is prepared by emulsifying a mineral oil such as vegetable oil or liquid paraffin as an oil phase and a natural acid phospholipid such as soy bean lecithin and an ester of anhydrous hyssitol or fatty acid such as sorbitan monooleate And hexylol anhydride such as dioxyethylene sorbitol monooleate, and condensation products of a partial ester derived from a fatty acid with ethylene oxide are emulsified to emulsify the active ingredient.

When the composition for external application for skin according to the present invention is formulated into a cosmetic, there is no particular limitation on the form of the composition, and there are no particular limitations on the formulation, and the softening lotion, convergent lotion, nutritional lotion, eye cream, nutritional cream, massage cream, cleansing cream, Water, powders, essences or packs.

Hereinafter, the present invention will be described in more detail with reference to the following test examples. However, these test examples are only intended to facilitate understanding of the present invention, and the scope of the present invention is not limited to these examples.

[Preparation Example 1] Preparation of 7,3 ', 4'-trihydroxyisoflavone

The 7,3 ', 4'-trihydroxyisoflavone was prepared by dissolving 3 g of daidzein (Sigma) in 100 ml of ionized water, sterilizing at 121 ° C for 30 minutes, cooling to 30 ° C, Bacillus subtilis KCCM 11732 was inoculated at 5-10% of the liquid amount. After 7 days of incubation at 37 ° C, the reaction was terminated. The culture solution was centrifuged at 5,000 to 10,000 rpm, and the collected precipitate was washed three times with distilled water and centrifuged again to obtain a precipitate. Ethanol (200 ml) was added to the precipitate and stirred (3 times), the precipitated salts were removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product. Then, the obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.45 g of 7,3 ', 4'-trihydroxyisoflavone.

[Test Example 1] PPARs activation effect

HaCaT, a human keratinocyte, was subcultured in DMEM medium containing 10% fetal bovine serum and phenol red-free medium was used to remove the effect of phenol red estrogen. Respectively. The plasmid is a promoter of the PPARs response element (PPRE), which is activated by binding of a plasmid having PPAR?, PPAR? /?, PPAR? Gene and ligand-binding PPARs after a universal promoter expressed under ordinary culture conditions. A plasmid in which a firefly luciferase gene is used as a reporter and a universal promoter to be used as a reference is coupled with a β-galactosidase gene [Clew, S. Kliewer, S. A.), Saturn, Rain. Forman, B.M., Blumberg, B., Ong, Lee. Ong, E. S., Borgmeyer, U., Mannelsdorf, D .; Mangelsdorf, D. J., Umesono, K., Evans, Al. Evans, R. M., Differential expression and activation of a family of murine peroxisome proliferator-activated receptors, Proc. Natl. Acad. Sci. USA, 91 (1994) 7355-7359.].

HaCaT cells were plated in a 24 well plate at a concentration of 5x10 < ⁴ > and cultured for 24 hours. Then, the plasmid gene was transiently transfected. After incubation for 24 hours, the cells were washed with phosphate buffered saline (PBS), and 7,3 ', 4'-trihydroxyisoflavone (3ODI) prepared in Preparation Example 1 and PPAR? Ligand Wy-14,643 and PPAR? The ligand, troglitazone (" TGZ "), was treated with 10 μM each. The negative control group was a group treated with ethanol used for dissolving the sample. Further, 7,6,4'-trihydroxyisoflavone (6ODI) and 7,8,4'-trihydroxyisoflavone (8ODI) having a structure similar to 7,3 ', 4'-trihydroxyisoflavone ) Were treated with 10 μM each to compare the PPARs activation effect. After incubation for 24 hours, the cells were washed with PBS, and the luciferase activity was measured by harvesting the cells. The results are shown in FIGS. 1A and 1B.

FIG. 1A shows the result of transfection of a PPAR promoter reporter plasmid and a PPARa expression plasmid into a HaCaT cell line, followed by treatment with a drug and measurement of luciferase activity. FIG. 1B shows the result of transfection of a PPAR promoter reporter plasmid and a PPAR? Expression plasmid into a HaCaT cell line, followed by treatment with a drug and measuring luciferase activity. The first column of each graph shows the luciferase activity of the ethanol treated group as a negative control, and the second column is the positive control group treated with Wy-14,643, the PPAR alpha ligand, and troglitazone, the PPAR gamma ligand, respectively. The calculated values of each treatment group were expressed as a ratio with the negative control group. As can be seen from FIG. 1A, the group treated with 7,3 ', 4'-trihydroxyisoflavone exhibited a high luciferase activity, leading to a significant degree of PPAR alpha activity. However, 7,6,4'-trihydroxyisoflavone and 7,8,4'-trihydroxyisoflavone were not effective in inducing PPAR [alpha] activity. As can be seen in FIG. 1B, the PPAR gamma transcriptional activity is similar to that of 7,3 ', 4'-trihydroxyisoflavone, 7,6,4'-trihydroxyisoflavone and 7,8,4'-trihydroxy Isoflavones were not affected by all three substances.

[Experimental Example 2] Modulating effect of PPARs, TGase 1, filaggrin and HAS2 gene expression

HaCaT was cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovine serum. 75 divides each 1x10 ^6, were incubated 24 hours in a flask. Thereafter, DMEM medium containing no fetal bovine serum was treated and cultured for 24 hours. Then, 7,3 ', 4'-trihydroxyisoflavone was added to the medium without fetal bovine serum for 24 hours Respectively. Cells were then harvested, washed with cold phosphate buffered saline (PBS), and 1 ml of TRIZol reagent (Life Technologies, Inc.) was added to extract the total RNA and quantitated by reverse transcription PCR was performed. Primer sequences of PPAR [alpha], [gamma], TGase 1, filaggrin, and HAS 2 for PCR are shown in Table 1 below.

4 쨉 g of total RNA was added to 25 쨉 l of a reverse transcription reaction buffer containing 50 mM Tris-HCl (Tris-HCl, pH 8.3), 75 mM KCl, 3 mM MgCl ₂ , 0.1 M DTT, 10 mM dNTP, 40 units / R RNase inhibitor, (DT) ₁₆ primer and a 200 unit SuperScript II (SuperScript II, GiboBRL) reverse transcription polymerase were added and reacted at 42 ° C for 1 hour. 2.5 μl of the reverse transcription reaction solution was then diluted with AmpliTaq DNA polymerase (0.04 U, Perkin Elmer, Shelton, CT), 50 mM Tris (pH 8.3), 0.25 mg / Was added to 50 L of a PCR reaction buffer containing 3 mM MgCl ₂ , 0.25 mM dNTPs and 1 / 50,000 dilution of SYBR Green I [Molecular Probes, Eugene, Ole (Molecular Probes, Eugene, OR)] and 10 μM of primer 30 C for 30 seconds, 53 C for 30 seconds, and 72 C for 1 minute.

Relative mRNA levels were analyzed by measuring changes in SYBR Green I fluorescence using ICycler software and are shown in FIG. The positive control group was Wy-14,643, which is a PPARα ligand, and TGZ, which is a ligand of PPARγ, and showed relative mRNA levels based on the value of 1 in the ethanol-treated group as a negative control.

The primer sequences used in PCR Gene / NCBI Gene ID Primer sequence order
number Amplification product size (bp) PCR reaction temperature (캜) denaturalization Annealing expansion β-actin / 5'-ATC CCA TCA CCA TCT TCC AG-3 ' One 579 94 58 72 5'-CCT GCT TCA CCA CCT TCT TG -3 ' 2 PPARα / 5'-ATG TCC GAA GCA AAC ATC AC-3 ' 3 401 94 58 72 5'-TAA TGT CCA GGA AGT AGG TG -3 ' 4 PPARy / 5'-TGC AGA GTTCCC CAA CTG GTA CAT C -3 ' 5 387 94 58 72 5'-GTG CTG CCT AAT GTC CCC TTG AAT C -3 ' 6 TGase1 / 5'-CCT GTA GCC CAC GTC GTA GC -3 ' 7 374 94 58 72 5'-TTG ACC TCA GCG CTG AGT TG -3 ' 8 Filaggrin / 5'-ACT CGG GGA TAA ATA GTT CCAA-3 ' 9 357 94 60 70 5'-CCT TAC ATA TAT ATT CCC CAG CAT-3 ' 10 HAS2 / 5'-TGA AGG ACC CTG CGA ACT TTC-3 ' 11 185 95 61 70 5'-GAA CAC TCG CTC AAA CCA GC-3 ' 12 5'-AGG TGT ATT GAC CCA TGC TAG AT-3 ' 16

FIGS. 2A to 2E are graphs showing the effect of 7,3 ', 4'-trihydroxyisoflavone according to the present invention on the expression levels of mRNAs of PPARα, PPARγ, TGase1, filaggrin and HAS 2. The first column of each graph shows the group treated with ethanol as the negative control group, and the second column treats Wy-14,643 and troglitazone as the positive control group, respectively. Wy-14,643 is a PPAR alpha ligand that has been reported to induce the differentiation of keratinocytes and to enhance the barrier. As can be seen from FIGS. 2A to 2E, the group treated with 7,3 ', 4'-trihydroxyisoflavone showed a significant increase in PPARα, PPARγ, filaggrin gene, transglutaminase 1 (TGase 1) Gene and the hyaluronic acid synthase 2 (HAS 2) gene. Although 7,6,4'-trihydroxyisoflavone and 7,8,4'-trihydroxyisoflavone also increased the expression of some genes, the 7,3 ', 4'-trihydroxy Compared with isoflavones, the effect was very small.

As described above, the 7,3 ', 4'-trihydroxyisoflavone according to the present invention induces PPARs transcription activity or increases expression thereof, thereby inducing differentiation of keratinocytes and thereby enhancing skin barrier, Moisturizing effect was increased. It also increases the expression of the filaggrin gene, a precursor protein of the Natural Moisturizing Factor (NMF) responsible for moisturizing the surface of the skin. Transglue, which plays a crucial role in the formation of the cornified envelope, Expression of hyaluronic acid synthase 2 (HAS2) gene, which is involved in the synthesis of hyaluronic acid, an extracellular matrix component that enhances the expression of Transglutaminase 1 (TGase 1) gene and performs skin moisture retention Of the total population. Accordingly, the present invention provides a composition for external application for skin barrier strengthening or moisturizing comprising 7,3 ', 4'-trihydroxyisoflavone as an active ingredient.

[Formulation Example 1] Nutritional lotion

Nutrition lotion was prepared according to a conventional method according to the composition shown in Table 2 below.

ingredient Content (% by weight) Purified water Balance glycerin 8.0 Butylene glycol 4.0 Hyaluronic acid extract 5.0 Beta Glucan 7.0 Carbomer 0.1 7,3 ', 4'-trihydroxyisoflavone 0.05 Caprylic / capric triglyceride 8.0 Squalane 5.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Cetearyl alcohol 1.0 Triethanolamine 0.1

[Formulation Example 2] Nourishing cream

Nutrition creams were prepared according to the compositions shown in Table 3 below in a conventional manner.

ingredient Content (% by weight) Purified water Balance glycerin 3.0 Butylene glycol 3.0 Liquid paraffin 7.0 Beta Glucan 7.0 Carbomer 0.1 7,3 ', 4'-trihydroxyisoflavone 3.0 Caprylic / capric triglyceride 3.0 Squalane 5.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Polysorbate 60 1.2 Triethanolamine 0.1

[Formulation Example 3] Massage cream

Massage creams were prepared in a conventional manner according to the composition shown in Table 4 below.

ingredient Content (% by weight) Purified water Balance glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 45.0 Beta Glucan 7.0 Carbomer 0.1 7,3 ', 4'-trihydroxyisoflavone 1.0 Caprylic / capric triglyceride 3.0 Wax 4.0 Cetearyl glucoside 1.5 Sesquioleic acid sorbitan 0.9 Vaseline 3.0 paraffin 1.5

[Formulation Example 4] Pack

The packs were prepared in a conventional manner according to the composition shown in Table 5 below.

ingredient Content (% by weight) Purified water Balance glycerin 4.0 Polyvinyl alcohol 15.0 Hyaluronic acid extract 5.0 Beta Glucan 7.0 Allantoin 0.1 7,3 ', 4'-trihydroxyisoflavone 0.5 Nonyl phenyl ether 0.4 Polysorbate 60 1.2 ethanol 6.0

[Formulation Example 5] Ointment

Ointments were prepared in a conventional manner according to the compositions shown in Table 6 below.

ingredient Content (% by weight) Purified water Balance glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 15.0 Beta Glucan 7.0 Carbomer 0.1 7,3 ', 4'-trihydroxyisoflavone 1.0 Caprylic / capric triglyceride 3.0 Squalane 1.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Cetearyl alcohol 1.0 Wax 4.0

FIGS. 1A and 1B show the results of confirming the PPARγ and PPARγ transcriptional activity of 7,3 ', 4'-trihydroxyisoflavone according to the present invention using PPARα and PPARγ reporter gene assay.

FIGS. 2A to 2E show the effect of the present invention on the expression levels of PPARα, PPARγ, TGase1, filaggrin and HAS2 mRNAs of 7,3 ', 4'-trihydroxyisoflavone according to the present invention in HaCaT cells using real-time reverse transcription DNA polymerization It is a graph showing the influence.

Claims (6)

A cosmetic composition for external application for skin for moisturizing, which comprises 7,3 ', 4'-trihydroxyisoflavone as an active ingredient, Wherein said 7,3 ', 4'-trihydroxyisoflavone activates (i) peroxisome proliferator-activated receptors ("PPARs"), (ii) 1 and the hyaluronic acid synthase 2 gene in the skin. A cosmetic composition for external skin application for skin barrier strengthening comprising 7,3 ', 4'-trihydroxyisoflavone as an active ingredient, Wherein said 7,3 ', 4'-trihydroxyisoflavone activates (i) peroxisome proliferator-activated receptors ("PPARs"), (ii) 1 < / RTI > and hyaluronic acid synthetase 2 gene of the present invention. The cosmetic composition for external application for the skin according to claim 1 or 2, wherein the 7,3 ', 4'-trihydroxyisoflavone is contained in an amount of 0.001 to 10% by weight based on the total weight of the composition. delete delete The composition for external application for skin according to claim 1 or 2, wherein the composition for external application for skin comprises at least one selected from the group consisting of softening agents, convergent lotion, nutritional lotion, eye cream, nutritional cream, massage cream, cleansing cream, cleansing foam, cleansing water, powder, essence, Wherein the cosmetic composition is an external preparation for skin.

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